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Journal of Bacteriology, September 2006, p. 6346-6353, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00462-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

General Mutagenesis of F Plasmid TraI Reveals Its Role in Conjugative Regulation

Rembrandt J. F. Haft,¹ Gilberto Palacios,¹ Tran Nguyen,¹ Manuela Mally,^2, Eliora G. Gachelet,¹ Ellen L. Zechner,² and Beth Traxler^1*

Department of Microbiology, University of Washington, Seattle, Washington 98195,¹ Institute of Molecular Biosciences, University of Graz, A-8010 Graz, Austria²

Received 3 April 2006/ Accepted 19 June 2006

Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies 100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.

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* Corresponding author. Mailing address: Box 357242, University of Washington, Seattle, WA 98195-7242. Phone: (206) 543-5485. Fax: (206) 543-8297. E-mail: btraxler{at}u.washington.edu.

Present address: Biozentrum, University of Basel, Molecular Microbiology, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.

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Journal of Bacteriology, September 2006, p. 6346-6353, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00462-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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